The introduction of the electron microscope made it necessary for the users thereof to dry the specimens that were going to be viewed through such a microscope. If such a specimen is not dried, the beam of electrons becomes difficult to focus; i.e., the electron beam is subjected to a moisture-contaminated "vacuum."
Originally specimens were simply air dried by exposing the specimen to the ambient air or to vacuum conditions, thus permitting the water in the specimen to evaporate. Such techniques were not entirely satisfactory since the surface tension of the water would cause a specimen to tear or be compressed during this form of drying procedure. Accordingly, a specimen so dried would wither and its three-dimensional characteristics would be distorted, thereby giving rise to a less than complete evaluation of a specimen held under an electron microscope.
At least one answer to overcoming the problem of having the specimens tear and become compressed was the introduction of the technique of critical point drying. The advantages of critical point drying were set out by T. F. Anderson in a paper entitled, "Techniques for the Preservation of Three-Dimensional Structure in Preparing Specimens for the Electron Microscope," published in the Transactions of the New York Academy of Science, Series II, 13, 130 (1951). In accordance with the critical point drying technique, the specimen containing water is first exposed (immersed) in a bath of ethanol. Thereafter the specimen is immersed in a bath of amyl acetate followed by being immersed in a bath of liquid CO.sub.2. The water is miscible with the ethanol; the ethanol is miscible with amyl acetate; and the amyl acetate is miscible with the liquid CO.sub.2. Accordingly, by diffusion the water is displaced by the ethanol, the ethanol is displaced by the amyl acetate, and the amyl acetate is displaced by the liquid CO.sub.2. The liquid CO.sub.2 is then changed to a gaseous state above the critical point so that all surface tension forces are eliminated during vaporization. By using this technique the specimen is dried, but it retains its original three-dimensional form.
The procedure described above is suitable for pieces of animal and plant tissue of reasonable size. However, no good method exists for containing such small particle materials as microorganisms or fibers. By small particle materials is meant those having diameters of from 1 to 1,000 microns. Bacteria, yeasts, and mold spores are often not larger than a few microns and are not easily contained during the critical point drying process without greatly prolonging the duration of the drying process.
The exchange of liquid during the displacement steps takes place from inside the biological cell to the surrounding medium. Such an exchange takes place by diffusion and the distances involved in the exchange are in the order of a few microns and the traveling times of the solvent molecules are in the order of 0.01 to 1 sec. The diffusion rate is directly related to the concentration gradient between the inside and outside of the cell membrane according to the equation Q = - A D P (.DELTA.C/.DELTA.x) t where Q = solute crossing a surface area A, D = diffusion coefficient, P = the permeability factor of the membrane, (.DELTA.C/.DELTA.x) = concentration gradient, and t = time. It has been determined that if the concentration gradient can be maintained at a high level by the flow of liquid through the sample container, the displacement time is greatly reduced from an ordinary critical point drying procedure where the rate of liquid exchange is determined by the slow process of diffusion. It has been determined that the concentration gradient can be held at a high level by agitation but in the technique employed heretofore, agitation has not been effected easily because small particle specimens to be dried have to be confined. The present device effects the high level of concentration gradient by continuous replenishment of the fluid coming in contact with the specimen.
It is known that microorganisms can be filtered from a fluid stream by passing the stream into a holder containing a filter disposed across the stream flow, thus collecting the microorganisms on the upstream surface of the filter. For example see U.S. Pat. No. 2,672,431.